Abstract

Human monocyte/macrophages (Mφ) exposed to nonparticulate stimuli can express cell surface Fas ligand (FasL) and release active soluble FasL (sFasL). We now report that monocyte/Mφ-ingesting opsonized zymosan released sFasL and conditioned supernatants so that these triggered Fas-mediated apoptosis of “bystander” monocytes and FasL-negative neutrophils. Furthermore, identical results were seen with Mφ taking up apoptotic neutrophils, whereas medium conditioned by Mφ phagocytizing latex beads had no proapoptotic effects upon neutrophils despite the presence of sFasL. These data suggest the hitherto unrecognized existence of a feedback loop requiring soluble factors in addition to sFasL that may promote resolution of inflammation-phagocytic clearance of apoptotic cells leading to Fas-mediated killing of bystander leukocytes by phagocytizing macrophages.

Cells of the monocyte/macrophage (Mφ)2 lineage are multifunctional, orchestrating many aspects of inflammation and tissue repair. A key property of the monocyte/Mφ, first recognized over a century ago by Metchnikoff (1), is the capacity to phagocytize particulate debris, invading microorganisms and effete cells. Indeed, there are now persuasive data to show that a critical event in resolution of inflammation is nonphlogistic safe phagocytic clearance of intact leukocytes undergoing deletion by apoptosis, also known as programmed cell death (2, 3, 4, 5, 6, 7, 8, 9, 10, 11).

In addition to clearing away apoptotic cells, important new data (12, 13) suggest that monocyte/Mφ can trigger apoptosis in other cells by regulated surface expression of Fas ligand (FasL) and by release of soluble FasL (sFasL). Since neutrophils, eosinophils, lymphocytes, and monocytes themselves all express the Fas “death receptor” and may, in certain circumstances, be susceptible to apoptosis triggered by cross-linking of Fas (12, 14, 15, 16, 17, 18), it has been suggested that monocyte/Mφ expression of FasL may be an important factor in regulating leukocyte populations at inflamed sites (12, 13). However, to date there has been little study of stimuli that control FasL expression/release by monocyte/Mφ, although there are reports that monocyte/Mφ FasL is up-regulated by nonparticulate stimuli such as soluble immune complexes, superantigen, phytohemagglutinin, and Ab-mediated cross-linking of monocyte/Mφ CD4 (13, 19, 20). In this report, we have studied monocyte/Mφ expression of FasL after phagocytosis of opsonized zymosan or apoptotic neutrophils, asking whether these stimuli induced release of sFasL from phagocytizing monocyte/Mφ and triggered Fas-mediated apoptosis in “bystander” neutrophils and monocytes.

Assessment of apoptosis

In the present study, monocyte and neutrophil apoptosis was routinely quantified by flow cytometry using FITC-conjugated annexin V (24) and using a Becton Dickinson FACScan (Oxford, UK), as previously described (25). We have extensively validated this technique against apoptosis as quantified by morphology on Giemsa-stained cytospins (3) and by shedding of CD16 (26), and we agree with published data (24) that, in myeloid cells, these different approaches yield closely comparable results (25).

Phagocytic stimulation of monocytes and macrophages

Mφ cultured in Costar plates (Wycombe, UK) or monocytes cultured in teflon-lined wells were coincubated with washed particles of OpsZ at a final concentration of 0.5 mg/ml for up to 7.5 h in Iscove’s medium in the absence of autologous serum. Typically 4 × 105 Mφ or 2 × 106 monocytes in a final volume of 400 μl were used per equivalent experiment. Aliquots of conditioned media were removed at times indicated and clarified by sequential centrifugation at 300 × g for 5 min to remove intact cells and cell debris and at 1200 × g for 5 min to remove all traces of OpsZ before being used to resuspend freshly isolated but untreated peripheral blood monocytes or neutrophils. Monocytes were resuspended to a density of 2 × 106/ml in teflon wells and incubated at 37°C for up to 19 h in the absence of autologous serum. Neutrophils, on the other hand, were cultured at 5 × 106/ml in the presence of 10% autologous PRPDS to undergo constitutive apoptosis without significant necrosis (3). In addition to OpsZ, 6-day Mφ were also incubated with latex beads at a final concentration of 0.5 mg/ml or with apoptotic neutrophils at 1 × 107/ml. When indicated, assays also contained the Fas antagonistic Ab ZB4 or an isotype-matched control negative Ab at an equivalent concentration.

FasL expression as determined by flow cytometry

Monocytes, maintained in the presence of 10% autologous PRPDS and cultured in teflon-lined “wells,” were assessed for FasL protein expression by indirect immunofluorescent labeling and flow cytometric analysis. Cells (1 × 105), resuspended in 100 μl of 10% newborn calf serum, were coincubated with 100 ng of an anti-FasL mAb, clone 33 (or a control isotype-matched mAb), at 4°C for 60 min. Cells were washed and recovered by centrifugation at 300 × g for 5 min before resuspending in 10% newborn calf serum containing an FITC-conjugated F(ab′)2 fragment of a sheep anti-mouse IgG polyclonal Ab. Cells were then analyzed by a Becton Dickinson FACScan flow cytometer for cell-associated fluorescence.

SDS-PAGE and protein blotting

Twelve percent SDS slab gels were prepared and run according to the method of Laemmli (27), with the exception that gels were ran with 1 mM thioglycollic acid added to the cathode buffer and with the anode buffer diluted twofold. Each sample was prepared on the basis of equal numbers of extracted cells rather than on protein content. A typical 100 × 100 × 1.5 mm gel was run at 25 mA for 2 h and transferred to polyvinylidene difluoride (PVDF) membranes at 2 mA/cm2 for 3 h at 4°C according to the methodology of Towbin et al. (28). Membranes were then blocked with 5% milk powder (Marvel, 99% fat-free) in PBS containing 0.2% Tween 20 for 1 h before probing overnight with either an anti-FasL mAb (clone 33) or pAb (Ab-1), with all steps maintained at 4°C. The presence of Tween 20 in buffers and maintaining immunoblotting conditions at 4°C were essential in minimizing cross-reactivity of clone-33 with β-actin (our unpublished observations). Blots were then washed with ice cold PBS before probing with a peroxidase-conjugated secondary pAb of appropriate specificity and detection with 4-chloro-1-naphthol.

SDS-PAGE and sample preparation

Serum-free media conditioned by neutrophils, monocytes, or Mφ were prepared for protein analysis by SDS-PAGE by first clarifying the supernatant at 500 × g, adding SDS to 0.1%, and immediately placing into a boiling water bath for 5 min. Aliquots (0.4 ml each) were then concentrated by centrifugation using Millipore Ultrafree filter units with a 10-kDa “cut-off.” The retentate was recovered with Laemmli sample buffer and boiled ready for SDS-PAGE analysis. The initial boiling of samples in the presence of 0.1% SDS was necessary for reproducible results, optimized with conditioned media taken from activated Jurkat T cells. Total cell protein was prepared by immediately resuspending cell pellets in hot Laemmli sample buffer and placing in a boiling water bath for 5 min.

Results

In keeping with earlier reports (16, 20), we were unable to detect surface expression of FasL by normal human peripheral blood monocytes prepared by elutriation of mixed mononuclear cells obtained from plasma-Percoll density gradient centrifugation of dextran-sedimented blood (Fig. 1⇓A). Furthermore, we were also able to confirm (13) that freshly isolated monocytes did not release FasL into supernatants during 3-h culture, as assessed by immunoblotting (Fig. 1⇓B). However, monocytes are believed to contain intracellular pools of FasL that can be rapidly mobilized to the cell surface by soluble stimuli, including immune complexes (13, 20). We examined whether uptake of opsonized zymosan (OpsZ), a well-established model of Ig/complement-mediated phagocytosis, might have similar effects. By 3 h, we observed increased surface expression of FasL by monocyte/Mφ and release of FasL into the supernatant by monocytes or monocyte-derived Mφ taking up OpsZ (Fig. 1⇓, A and B).

Phagocytosis of OpsZ stimulates monocyte/Mφ expression of surface FasL and release into supernatants. A, Monocytes, cultured in teflon-lined “pools” for up to 3 days in PRPDS, were immunolabeled for FasL cell surface expression with the mAb clone 33 and detected with a FITC-conjugated F(ab′)2 sheep anti-mouse polyclonal Ab before analysis by flow cytofluorometry. Control mAb, matched for isotype (IgG1) and quantity, was essentially superimposable with an autofluorescent (unlabeled) control and confirmed the absence of nonspecific binding. Fresh monocytes were also incubated for 3 h in suspension with OpsZ at 0.5 mg/ml. These results are representative of three separate experiments performed with different donors each time. B, Soluble FasL was not detected in the conditioned media of unstimulated monocytes (lane i, n = 9 experiments) or Mφ (lane ii, n = 6) but was easily demonstrable following ingestion of OpsZ (lane iii, n = 6, and lane iv, n = 6, respectively). Controls were supernatants conditioned by Jurkat T cells stimulated with A23187/TPA (lane v; showing release of FasL) or cultured in medium alone (lane vi). Conditioned supernatants were taken from 3-h cultures and processed for SDS-PAGE as described in Materials and Methods, with transblots probed for FasL using the mAb Clone 33. Molecular mass markers (S) were from Sigma (SDS-7B).

Freshly isolated monocytes cultured in the absence of serum are susceptible to apoptosis induced by Fas ligation (12, 14, 17, 28). Preliminary experiments (data not shown) indicated that supernatants freshly obtained from monocyte/Mφ taking up OpsZ could induce apoptosis in “target” monocyte populations; a supernatant transfer approach was taken in preference to coculture to avoid underestimation of apoptosis in the target population because of phagocytic removal of apoptotic cells by Mφ. Unfortunately, the proapoptotic effects of media conditioned by monocyte/Mφ-ingesting OpsZ were poorly preserved by freezing or storage at 4°C, despite no significant changes in sFasL protein as assessed by Western blot analysis (data not shown). Therefore, we designed an experiment (Fig. 2⇓A) in which two populations of monocytes (“donor” and “target”) were prepared at the same time from any given donor. As previously reported (29), monocytes in the target population cultured in the absence of serum underwent apoptosis (∼30% at 19 h), and this could be partially inhibited by the function-blocking Fas mAb ZB4 (Fig. 2⇓A, left bars), in keeping with data implicating Fas/FasL-mediated fratricide in such cultures (12). However, when supernatants conditioned by donor monocytes ingesting OpsZ were transferred to target monocytes at the end of the conditioning period (either 2.5 h or 7.5 h), increased apoptosis in the target monocytes was observed when the experiment was terminated at 19 h after cell isolation (Fig. 2⇓A, center and right bars). With increasing conditioning time the proapoptotic effect of supernatants from monocytes ingesting OpsZ increased. Nevertheless, stimulated apoptosis of target monocytes was partially abrogated by Fas blockade with ZB4. Supernatants conditioned by donor monocytes cultured in medium alone had no proapoptotic effect (Fig. 2⇓A, right bar). Essentially similar results were obtained when 3-day monocyte-derived Mφ were employed as the donor population (Fig. 2⇓B), except that medium conditioned by Mφ cultured without OpsZ protected monocytes against constitutive apoptosis (Fig. 2⇓B, right bar).

Fas-mediated death of monocytes is differentially promoted with supernatants from monocytes and macrophages ingesting OpsZ. Fresh monocytes (A) or 3-day Mφ (B) were incubated with OpsZ for the indicated conditioning times, and the media were removed, clarified, and either control IgG (gray bars) or ZB4 (white bars) were added to a final concentration of 1200 ng/ml. Clarified supernatants were then used to resuspend and incubate fresh peripheral blood monocytes at 2 × 106/ml in culture for up to 19 h. Apoptosis was assessed by annexin-V labeling and confirmed by Giemsa-stained cytospins. The black bars represent conditioned media controls for untreated monocyte or 3-day Mφ cultures.

These data strongly suggested that FasL and/or related molecules released from monocyte/Mφ phagocytizing OpsZ accounted for the proapoptotic effect of conditioned medium. However, the possibility remained that some other factor might have up-regulated Fas/FasL-mediated fratricide in the target monocyte population. Therefore, we went on to seek target cells of relevance to the inflammatory response that did not exhibit Fas/FasL fratricide.

Supernatants from monocyte/Mφ-ingesting OpsZ also induced Fas-mediated death in FasL-negative neutrophils

Neutrophils are widely recognized to express Fas and to be moderately sensitive to apoptosis induced by Fas ligation (14). We confirmed that relatively high concentrations of the agonistic Fas mAb CH-11 were able to accelerate constitutive apoptosis in highly purified neutrophils prepared from normal human blood by plasma-Percoll density gradient centrifugation (Fig. 3⇓A). However, by contrast with monocytes prepared by the same techniques (Fig. 2⇑), there was no evidence of Fas/FasL-mediated fratricide, in that Fas blockade with mAb ZB4 had no effect on constitutive apoptosis of neutrophils. This was despite the ability of ZB4 being able to block CH-11-induced apoptosis (Fig. 3⇓A). Furthermore, ZB4 did not inhibit constitutive apoptosis in neutrophils cultured in the absence of serum (data not shown), indicating that serum was not masking Fas/FasL-mediated fratricide.

Constitutive apoptosis in highly purified populations of neutrophils is not Fas mediated. A, Isolated senescent neutrophils cultured in teflon “pools” constitutively undergo apoptosis over a 24-h period as assessed morphologically from Giemsa-stained cytocentrifuge preparations. The Fas antagonistic Ab ZB4 was added to freshly isolated neutrophils and in some experiments (two groups of three bars on right of panel) was followed 30 min later by the addition of the Fas agonistic Ab CH-11. All experiments were controlled with an isotype matched Ab (IgM for CH-11; IgG1 for ZB4); n, The number of replicate experiments with different blood donors; error bars are the 95% confidence interval by unpaired Student t test. Note that ZB4 at 500 ng/ml has no effect on constitutive apoptosis but completely blocks increased apoptosis induced by CH-11. B, Total cell protein from the equivalent of 3 × 106 cells for freshly isolated (lane ii) and apoptotic (lane iii) neutrophils, in addition to the deliberate activation of fresh neutrophils by adherence to plastic (lane iv), FMLP (50ng/ml)/LPS (1 μg/ml) (lane v), or phagocytosis of OpsZ (lane vi), was separated by SDS-PAGE, and transblots were probed with the anti-FasL mAb clone-33. Extracts from the equivalent of 1 × 105 Jurkat T cells are shown as a positive control (lane i). Molecular mass markers (S) were from Sigma (SDS-7B). Identical results were obtained with a polyclonal Ab to FasL (Ab-1), as well as with immunoprecipitates prepared from 50 × 106 cells (data not presented) lysed with 1% Triton X-100 in 50 mM HEPES buffer containing 1 mM 1,10-phenanthroline, 1 mM EDTA, 1 mM PMSF, 10 μM leupeptin, 10 μM antipain, and 10 μM pepstatin. Ingestion of OpsZ by monocytes (C) and Mφ (D) induced Fas-dependent neutrophil apoptosis. Clarified conditioned media containing a control IgG (gray bars) or ZB4 (white bars) mAb at 1200 ng/ml were used to resuspend freshly isolated PMNs to 5 × 106/ml in 10% autologous PRPDS. The black bars represent conditioned media controls for untreated monocyte or 3d-MDMφ cultures, and error bars represent the 95% confidence interval.

In addition, we were unable to detect FasL protein by neutrophils from 16 different healthy donors using techniques simultaneously proven to reveal FasL expression by activated Jurkat T cells or stimulated/matured monocytes (Fig. 3⇑B). Furthermore, we were unable to detect sFasL in the supernatants of highly purified neutrophil preparations (data not shown), important evidence against the possibility that failure to detect FasL might reflect cleavage from the cell surface. Previous reports that have used different granulocyte isolation protocols (16, 20) suggest that neutrophils may express FasL, in a manner that may be dependent upon techniques of preparation (20). However, no FasL expression could be detected by immunoblotting of highly purified neutrophils subjected to deliberate activation by adherence to plastic, exposure to various concentrations of FMLP and LPS, or phagocytosis of OpsZ (Fig. 3⇑B), data independently confirmed by flow cytometry and where the presence or absence of serum (human or bovine) had no effect (data not shown). Furthermore, medium conditioned by cultured neutrophils that possessed no sFasL by Western blot analysis had no proapoptotic effect when used to resuspend neutrophils, monocytes, or Jurkat T cells (data not shown). Nevertheless, it was possible to detect FasL in neutrophil lysates/culture supernatants when these were deliberately contaminated by as few as 1% monocytes (not shown), evidence against the possibility that degradation of FasL by neutrophil proteases might mask FasL expression.

When highly purified, FasL-negative neutrophils were employed as a “target” population, essentially similar results to those observed with monocyte targets were obtained: medium conditioned by “donor” monocyte/Mφ taking up OpsZ accelerated constitutive apoptosis in target neutrophils in a manner that was partially inhibitable with Fas-blocking ZB4 (Fig. 3⇑, C and D; compare with Fig. 2⇑). Again, supernatants conditioned by donor Mφ not receiving OpsZ had a protective effect (Fig. 3⇑D). These data suggested that monocyte/Mφ might have an important role in regulating apoptosis in neutrophils that have also been summoned to inflammatory sites. Since there is now compelling evidence that granulocytes undergoing apoptosis at inflamed sites are phagocytically cleared by monocyte/Mφ (3, 4, 5, 7, 8), we went on to examine whether Mφ ingestion of apoptotic neutrophils might also promote release of FasL.

“Donor” monocyte-derived Mφ were cocultured with various particulate stimuli for 4 h, the medium was harvested and clarified, and then it was incubated with freshly isolated neutrophils for 15 h, after which time apoptosis in the “target” neutrophils was assayed (Fig. 4⇓). Mφ taking up apoptotic neutrophils not only released FasL into the supernatant but also yielded conditioned medium that accelerated neutrophil apoptosis to a degree almost completely inhibitable by Fas-blocking ZB4 (Fig. 4⇓ (inset), lane iv). However, no proapoptotic effect was observed with medium conditioned by Mφ cultured for 4 h with freshly isolated neutrophils that are not taken up (2, 3), or by Mφ-ingesting latex beads. However, although fresh neutrophils did not incite FasL release by Mφ (Fig. 4⇓ (inset), lane ii), uptake of latex beads did cause FasL release (Fig. 4⇓ (inset), lane iii), indicating that additional Mφ-derived factors are required to contribute to the Fas-mediated proapoptotic effects of Mφ-conditioned medium.

Apoptotic and not fresh neutrophils stimulate Mφ to release bioactive FasL. Conditioned media taken from 6-day Mφ cocultured for 4 h with media alone (i), fresh neutrophils at 10 × 106/ml (ii), latex beads at 0.5 mg/ml (iii), apoptotic PMNs at 10 × 106/ml (iv), or OpsZ at 0.5 mg/ml (v), all supplemented with 10% PRPDS, were clarified, and an IgG1 control mAb (unshaded bars) or ZB4 (shaded bars) at 1250 ng/ml was added. These conditioned media samples were then used to resuspend freshly isolated neutrophils at 5 × 106/ml, and neutrophil apoptosis was determined 15 h later by FACS analysis of annexin-V-labeled cells. Also shown (extreme left) is the fresh media control for neutrophils where no exchange with culture media from 6-day Mφ was used. Error bars represent the 95% confidence interval for n = 5 experiments, each done in duplicate. Inset, The conditioned supernatants from 6-day Mφ were also analyzed by Western blot for FasL with molecular mass markers (S) from Sigma Chemical Co. (SDS-7B) indicated. The band recognized in the far right lane (S) is contamination from (v) at the time of sample loading.

Discussion

The experiments described above add importantly to previous work implicating Fas and FasL in myeloid leukocyte homeostasis (12, 13, 14, 16, 17, 30). Using a supernatant transfer approach, the data demonstrate that monocyte/Mφ-ingesting opsonized zymosan, a model of phagocytosis of Ab and complement-coated particles, releases FasL and triggers Fas-mediated apoptosis in “target” neutrophils. Furthermore, identical results were seen with Mφ taking up apoptotic neutrophils, whereas medium conditioned by Mφ-ingesting latex beads had no proapoptotic effect.

The in vivo relevance of these data needs very cautious interpretation, but the findings suggest a hitherto unrecognized role for phagocytizing macrophages in directing neutrophil (and monocyte) elimination from inflamed sites. It is particularly intriguing that the data point to a new feedback loop promoting resolution of inflammation (phagocytic clearance of apoptotic neutrophils by macrophages causes release into the microenvironment of factors triggering accelerated removal of neutrophils by Fas-mediated apoptosis), which not only down-regulates the injurious potential of this cell but also targets the neutrophil for injury-limiting phagocytic removal (3, 31, 32). Indeed, our data support reports that Mφ-ingesting apoptotic cells may generate antiinflammatory responses such as IL-10 release (33, 34), because Mφ-derived FasL might be expected to promote “antiinflammatory” elimination of other leukocytes, including eosinophils, monocytes, and activated T cells (8, 11, 13). A further speculation of interest is that a similar negative feedback might occur when Mφ take up particles (such as bacteria) coated with Ig and complement.

However, it is important to note that the proapoptotic effects of medium conditioned by phagocytizing monocyte/Mφ appear to be only partially mediated by ligands of Fas, since mAb ZB4-mediated blockade of Fas on target monocytes and neutrophils was not completely protective. Indeed, the probable production by phagocytizing Mφ of factors that sensitize target cells to FasL, or possible Mφ release of alternative ligands for Fas, is emphasized by the observation that supernatants obtained from Mφ-ingesting latex beads were not proapoptotic for neutrophils, despite containing FasL demonstrable by immunoblot. Clearly, further work would be required to characterize such factors, which preliminary work suggests are unstable at 4°C but likely to be of 10 kDa or greater.

These findings add to data indicating that various inflammatory mediators may regulate susceptibility of myeloid cells to Fas-mediated apoptosis (28) and are not inconsistent with recent reports suggesting that soluble FasL cleared from cell surfaces may not, in certain circumstances, promote apoptosis in “target” cells expressing Fas (35). Clearly, future studies will need to clarify the mechanisms by which monocytes/Mφ release FasL and related proapoptotic factors.

The current data clearly indicate that highly purified neutrophils did not express FasL at levels sufficient to be detected by immunofluorescence and immunoblotting or to induce Fas/FasL-mediated fratricide in culture. Preliminary RT-PCR experiments, employing appropriate positive controls, confirm lack of FasL mRNA expression by neutrophils (F. Salway and S. Brown, unpublished data). Different experimental conditions, as suggested by the studies of Mincheff et al. (20), could explain the apparent discrepancy between the current data and those of Liles et al. (16), particularly since that group’s own work demonstrated that, under some conditions, freshly isolated monocytes did not express FasL (16) whereas, under others, they did (12, 13). Furthermore, a recent report (36) suggests that the polyclonal Abs used in these later studies (12, 13) may be inappropriate for probing cell surface FasL expression by flow cytometric analysis (36). As for Western blot analysis, it may be pertinent that, in our experiments, neutrophil populations contaminated with as little as 1% mononuclear cells appeared to express FasL, a finding that also indicates that we were unlikely to be missing a possible intracellular pool of FasL in neutrophils (20). We conclude from the available data that, while FasL is not expressed at detectable levels by neutrophils under the conditions employed in this study, this does not discount the possibility of FasL expression under other conditions. However, debate over whether neutrophils are truly able to indulge in Fas/FasL-mediated fratricide may be somewhat academic, in that acutely inflamed tissues are rapidly infiltrated by Fas-bearing neutrophils and by monocytes, which are generally agreed to be capable of FasL expression.

Finally, the data also demonstrate that cultured unstimulated Mφ secrete factors that suppress apoptosis in both monocytes and neutrophils, consistent with previous data indicating that cytokines known to be elaborated by Mφ, such as granulocyte-macrophage (GM)-CSF, can inhibit apoptosis in both cell types (29, 37). These findings serve only to emphasize the potentially central importance of the macrophage in regulating elimination from inflamed sites of other leukocytes by apoptosis.

To conclude, our studies demonstrate that monocyte/Mφ taking up OpsZ or apoptotic neutrophils release FasL and promote Fas-mediated apoptosis of monocytes and neutrophils. This may represent a hitherto unrecognized negative feedback loop serving to promote resolution of inflammation by accelerating deletion of leukocytes by apoptosis.